NPY-mediated inhibition of neurally evoked contraction in the dog urinary bladder and localisation of NPY receptor subtype mRNA expression

Suzanne Dowson, Sheridan Piper-Brown, Rachel McCoy & Clare Christy, PGRD, Ramsgate Road, Sandwich, Kent CT13 2NJ.

Neuropeptide Y (NPY) is a neuromodulatory peptide found in the central and peripheral nervous system which mediates its actions via six receptor subtypes (Y1-Y6). NPY is implicated in multiple physiological processes and is abundant in bladder smooth muscle of several species, including human, where it is colocalised with adrenergic and cholinergic neurons. Whilst NPY has been shown to inhibit neurally-evoked contraction of bladder strips from the rat, regional differences in NPY activity within the bladder and the receptor subtypes involved in this process have not been thoroughly investigated to date.

The present study aimed to (i) characterise the neurotransmitters contributing to neurally-evoked contraction of dog bladder (ii) investigate NPY-mediated neuromodulation of the bladder and (iii) localise NPY receptor mRNA expression in the lower urinary tract.

All experiments were carried out in compliance with UK legislation. Urinary bladder and urethra from euthanised male Beagle dogs (10-15kg) were isolated and longitudinal smooth muscle strips dissected from bladder body, trigone and bladder neck. Isometric tension recordings were performed from strips bathed in Krebs’ solution (37°C, 95% O2 / 5% CO2) containing propranolol (1 μM), doxazosin (0.1 μM) and naproxen (10 μM). Strips were equilibrated (resting tension 1 g) for 1hr before electrical field stimulation (EFS)-induced contractions were evoked at 5 minute intervals (optimal parameters for bladder body, trigone and bladder neck were established in study). The neurotransmitter contributions to contraction were determined and inhibition of EFS-evoked contraction by NPY receptor agonists were subsequently investigated (N=4-6). Functional data are expressed as mean percentage maximum response or EC50 values (geometric mean) with 95% confidence intervals, N=4-6. Statistical analysis was carried out by Student’s t-test, with p<0.05 considered significant. NPY Y1, Y2, Y4 and Y5 mRNA expression in bladder and urethra was determined by qRT-PCR using the delta CT method (n=2).

EFS-evoked contractions of bladder strips were mediated by acetylcholine (45-73%; lower contribution in the trigone and bladder neck than bladder body (p<0.01)), noradrenaline (13-29%; greater contribution in trigone and bladder neck than in bladder body (p<0.01 and p<0.05 respectively)) and ATP (5-8%). Concentration-dependent inhibition of EFS-evoked contractions by hNPY, pNPY, PYY, and pNPY(13-36) were observed in the bladder neck (EC50 0.67nM (0.31-1.45), 0.49nM (0.25-0.98), 0.12nM (0.10-0.43), and 0.13nM (0.07-0.23) respectively; Emax ~55% for all) and bladder body (EC50 0.33nM (0.20-0.54), 0.20nM (0.11-0.38), 0.07nM (0.05-0.09), and 0.24nM (0.14-0.41) respectively; Emax 67%, 51%, 52% and 40% respectively). Agonist effects in the trigone were not significantly different from time-matched controls. NPY receptor mRNA was detected in bladder body (Y1 > Y5), trigone (Y1>Y2 =Y5), bladder neck (Y1>Y2 =Y5), and proximal urethra (Y1>Y2 =Y4=Y5).

The data presented support a role for NPY and its receptors in the neuromodulation of bladder function.